FROM IN VITRO TO IN VIVO: CONTROL OF C-REACTIVE PROTEIN GENE EXPRESSION BY CYTOKINES by DUPRANE PEDACI YOUNG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisors: Dr. David Samols & Dr. Irving Kushner Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ________________Duprane Pedaci Young___________________ candidate for the _____Doctor of Philosophy__________degree *. (signed)_______Hung-Ying Kao, Ph.D._____________________ (chair of the committee) ______________Irving Kushner, M.D._________________ ______________Alan D. Levine, Ph.D.________________ _______________Yu-Chung Yang, Ph.D.______________ _______________David Samols, Ph.D.________________ ________________________________________________ (date) ________1/22/08_______ *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION This thesis is dedicated to my husband, Greg, and my daughter, Cora. iii TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………...… vi ACKNOWLEDGEMENTS…………………………………………………………. viii LIST OF ABBREVIATIONS…………………………………………………….…... ix ABSTRACT…………………………………………………………………….…….. 1 CHAPTER 1: INTRODUCTION…………………………………………………… 3 The Acute Phase Response (APR)………………………………………………….…. 3 Regulation of Acute Phase Protein Gene Expression………………………….………. 5 C-Reactive Protein……………………………………………………………………. 16 Regulation of C-Reactive Protein Gene Expression During Cellular Stress…….…… 22 Regulation of C-Reactive Protein Gene Expression During the APR………….....….. 24 CHAPTER 2: THE INTERACTION OF C-REL WITH C/EBPβ ENHANCES C/EBPβ BINDING TO THE C-REACTIVE PROTEIN GENE PROMOTER… 34 INTRODUCTION………………………………………………………….………… 34 MATERIALS & METHODS………………………………………………………… 37 Materials……………………………………………………………………… 37 Plasmid Constructs…………………………………………………………… 38 Protein Purification…………………………………………………………… 39 EMSA………………………………………………………………………… 41 Data Analysis…………………………………………………………………. 42 Immunoprecipitation and Western Analysis………………………..………… 43 Cell Culture, Transient Transfection, and Luciferase Transactivation Assay… 44 iv RESULTS……………………………………………………………………………... 46 C/EBPβ Binding to a Consensus Oligo and to the CRP-C/EBP(-53) Oligo….… 46 Rel Family Members Binding to the CRP-κB(-43)B Oligo……………………… 52 c-Rel Enhances the Binding of C/EBPβ on the CRP-C/EBP(-53) Oligo……..… 58 DISCUSSION…………………………………………………………………………. 72 CHAPTER 3: BINDING OF C/EBPβ TO THE CRP PROMOTER IN HEP3B CELLS IS ASSOCIATED WITH TRANSCRIPTION OF CRP mRNA……….… 76 INTRODUCTION……………………………………………………………..….…… 76 MATERIALS & METHODS………………………………………………………….. 80 Materials………………………………………………………………...……… 80 Cell Culture and Cytokine Treatment…………………………………..……… 81 Chromatin Immunoprecipitation Assays……………………………….……… 81 Data Analysis…………………………………………………………..………. 83 RNA Isolation and RT-PCR……………………………………………..…….. 83 Whole Cell Extraction, SDS-PAGE and Immunoblot………………....………. 84 RESULTS…………………………………………………………………….….…….. 85 DISCUSSION………………………………………………………………..……….. 105 CHAPTER 4: SUMMARY…………………………………………….…………… 110 DISCUSSION….…………………………………………………………….………. 110 FUTURE DIRECTIONS…………………………………………………….……….. 114 REFERENCES…..………………………………………………………….……….. 120 v LIST OF FIGURES Figure 1: C/EBPβ and STAT3 protein domain maps and STAT3 signaling pathway………………………………………………………………………. 9-10 Figure 2: Domain maps for NF-κB and IκB families of proteins………………..…. 11-12 Figure 3: Classical NF-kB Activation Pathway……………………….………….… 13-14 Figure 4: Co-crystal structure of CRP with phosphocholine……………………..…. 18-19 Figure 5: Model of the proximal CRP promoter and relevant transcription factors… 26-27 Figure 6: Protein preparations and sequences of oligonucleotides used in EMSAs... 48-49 Figure 7: C/EBPβ exhibits a 60 fold higher binding affinity for a C/EBP consensus oligo than for the CRP-C/EBP(-53) oligo………………………… 50-51 Figure 8: p50 homodimer binding to the CRP-κB(-43)B site has an affinity comparable to that for the Ig-κB site and exhibits cooperative binding to the CRP κB(-43)B site……………………………………………………… 54-55 Figure 9: Pattern of binding of Rel Family Members to CRP-κB(-43)B and Ig-κB is similar……………………………………………………………………… 56-57 Figure 10: c-Rel(1-300) but not p50 or p52 enhanced C/EBPβ DNA binding on the CRP promoter………………………………………………………..……. 60-61 Figure 11: c-Rel(1-300) increased Kapp of C/EBPβ for CRP-C/EBP(-53) more than 10 fold………………………………………………………….………….. 62-63 Figure 12: c-Rel(1-300) physically interacts with C/EBPβ in solution……………… 66-67 vi Figure 13: The CRP-κB(-43)B site is not required for the enhancing effect of c-Rel(1-300) on C/EBPβ binding to the CRP promoter……………….……… 68-69 Figure 14: Overexpressed c-Rel(1-300) in combination with overexpressed C/EBPβ transactivates the CRP promoter………………………………...………….. 70-71 Figure 15: Current model of the proximal CRP promoter and relevant transcription factors…………………………………………….…………………………. 78-79 Figure 16: C/EBPβ, NF-κB p50, STAT3, c-Rel, and TBP bind the endogenous CRP promoter…………………………………………………………………….. 87-88 Figure 17: C/EBPβ binds the endogenous CRP promoter in response to Cytokines………………………………………………………………….… 89-90 Figure 18: p50 occupancy of the CRP promoter remains nearly constant in the presence or absence of cytokines……………………………………….…… 91-92 Figure 19: STAT3 occupancy of the CRP promoter rises modestly in response to cytokines……………………………………………………………………. 93-94 Figure 20: c-Rel and TBP occupy the CRP promoter in parallel…………………… 97-99 Figure 21: CRP mRNA accumulates in response to cytokines……………….….. 100-101 Figure 22: C/EBPβ and c-Rel protein levels increase in response to cytokine treatment………………………………………………………………….. 103-104 Figure 23: Theoretical model of kinetics of transcription factor occupancy on the CRP promoter before and after cytokine induction…………………….… 112-113 vii ACKNOWLEDGEMENTS I would like to thank my advisors Dr. David Samols and Dr. Irving Kushner for their training, guidance, and support. Dr. Samols taught me how to design and implement experiments and how to present scientific data in a clear and concise manner. Dr. Kushner taught me how to write logically, precisely, and clearly. Both Dr. Samols and Dr. Kushner helped me to become an independent scientist. My other committee members, Dr. Hung-Ying Kao and Dr. Alan Levine were extremely helpful throughout my graduate career. Dr. Kao and his students helped me with a number of assays and allowed me to use a variety of equipment for my experiments. Dr. Levine provided excellent constructive criticism during all of my committee meetings and helped steer my project along its final path. I thank Sui Bi Samols for teaching me how to perform a ChIP assay and for helping me optimize the ChIP assay. Hong Zhang taught me how to do real-time PCR and RT-PCR and was always available to discuss experimental ideas and conditions. Kris Stanya and Erin Reinecke both taught me how to perform several assays and were also available to discuss various experimental details and problems. I thank Dr. Richard Hanson for his kind recommendations and assistance with obtaining a postdoctoral position. I would also like to thank Dr. G.J. Darlington for providing Hep3B cells. This work was supported by National Institutes of Health grant #AG02467, the Metabolism Training Program grant #T32 DK-007319-28, and the Department of Biochemistry. viii LIST OF ABBREVIATIONS APP, acute phase protein APR, acute phase response CAT, chloramphenicolacetyl transferase C/EBP, CAAT/enhancer binding protein ChIP, chromatin immunoprecipitation CREBH, cAMP responsive element binding protein H (a.k.a. CREB 3 like protein 3) CRP, C-reactive protein EMSA, Electrophoretic Mobility Shift Assay Hep3B, hepatoma 3B HNF, Hepatocyte Nuclear Factor IFN γ, Interferon γ IL, interleukin Kapp, apparent equilibrium binding constant LAP, liver-enriched activating protein LIP, liver-enriched inhibitory protein NF-κB, Nuclear Factor κB STAT, Signal Transducer and Activator of Transcription TBP, TATAA Binding Protein TGF β, Transforming Growth Factor β TNF α, Tumor Necrosis Factor α UPR, unfolded protein response UPRE, unfolded protein response element ix From in vitro to in vivo: Control of C-Reactive Protein Gene Expression by Cytokines Abstract by DUPRANE PEDACI YOUNG Expression of the acute phase protein C-reactive protein (CRP) is tightly regulated in hepatocytes. While very little CRP mRNA is transcribed normally, inflammatory stimuli are followed by a dramatic increase in mRNA synthesis and accumulation. Interleukins -6 and 1β (IL-6 and IL-1β) are believed to be the major cytokines responsible for induction of CRP and other acute phase proteins. We previously demonstrated that in vitro c-Rel plays a novel regulatory role by forming a complex with C/EBPβ when C/EBPβ is bound to the CRP gene promoter following cytokine stimulation. c-Rel does not by itself bind to the DNA. In these studies we found that recombinant c-Rel(1-300) (lacks transactivation domain) increased the affinity of recombinant C/EBPβ for a CRP-derived C/EBP site (-53) at least 10 fold. C/EBPβ and c-Rel(1-300) were found to physically interact in solution, and overexpression of c-Rel in the presence of overexpressed C/EBPβ stimulated CRP transcription. We concluded that c-Rel(1-300) binding to C/EBPβ increased the affinity of C/EBPβ for the CRP-C/EBP(-53) site, and that the transactivation domain of c-Rel is not necessary for this effect, which depends on protein: protein contacts